Estandarización del método PCR para la identificación genotípica de Raoultella planticola, Raoultella ornithinolytica y Klebsiella oxytoca en muestras remitidas al (CRN RAM) INSPI Quito

Abstract

Bacterial multidrug resistance represents a serious problem for public health at a national and international level, due to the pathogenicity present in some enterobacteria and the repercussions they may have. The correct management, control and treatment of infections caused by these microorganisms are preceded by efficient identification. Various studies report difficulties in differentiating species belonging to the genera Raoultella and Klebsiella, due to the similarity in taxonomic and phylogenetic characteristics that they share. Phenotypic assays are insufficient, so the information is counteracted with genotypic methods based on the amplification of specific regions. The present study proposed the standardization of a PCR protocol for the identification of Raoultella planticola, Raoultella ornithinolytica and Klebsiella oxytoca based on the detection of the bla, hdc, pehX genes in strains from various hospital centers that are part of the national health system. For this, optimal thermal conditions were established in PCR, adequate concentrations of primers, and volumes of the components for the reaction. The protocol was able to correctly identify the microorganisms of interest based on the amplification of the genes bla and hdc in Raoultella ornithinolytica, hdc in Raoultella planticola and pehX in Klebsiella oxytoca. The optimal thermal amplification conditions were: Initial denaturation for 5 minutes at 95 °C, followed by 30 cycles of: denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds and extension at 72 °C for 1 minute each, final extension at 72°C for 7 minutes and maintenance of the DNA at 4°C indefinitely. The optimal concentration of primers for the reaction was 0.6uM.